TTF-1过表达抑制肺腺癌A549细胞增殖和上皮-间质转化

Overexpression of TTF-1 inhibits proliferation and epithelial-mesenchymal transformation of lung adenocarcinoma A549 cells

目的探讨甲状腺转录因子-1(TTF-1)基因过表达能否抑制肺腺癌A549细胞增殖和上皮-间质转化(EMT)。方法将TTF-1基因的编码序列(CDS)克隆至pCDH-CMV-MCS-EF1-CopGFP-T2A-puro慢病毒载体,构建TTF-1过表达慢病毒载体,在293T细胞内包装为TTF-1基因过表达的慢病毒颗粒,转染至肺腺癌细胞A549;用iCelligence实时无标记细胞功能分析仪检测TTF-1转染组(LV-TTF1)、空载体组(vector)及未转染组(NC)3组A549细胞增殖水平;3组细胞分别予TGF-β1刺激后,观察细胞的形态变化,RT-qPCR法检测EMT相关标记物表达水平的改变,细胞划痕及Transwell迁移实验法检测细胞转移能力的变化。结果RT-qPCR及Western Blot结果显示,成功构建TTF-1过表达A549肺腺癌细胞系;iCelligence结果显示,与vector、NC组相比,LV-TTF1组细胞增殖水平降低;LV-TTF1组细胞经TGF-β1刺激后,未发生明显的EMT形态学变化,而vector、NC组细胞形态变化明显;LV-TTF1组细胞中E-cadherin mRNA表达量较NC明显上升(P<0.05),Vimentin mRNA的表达明显降低(P<0.05);划痕实验中LV-TTF1组的细胞迁移率<NC组(P<0.05),Transwell迁移实验中LV-TTF1组细胞迁移数量明显少于NC组(P<0.05)。结论慢病毒载体介导TTF-1过表达能够抑制肺腺癌A549细胞增殖和EMT。

Objective To investigate whether overexpression of TTF-1 can inhibit the proliferation and epithelial-mesenchymal transition(EMT) of lung adenocarcinoma A549 cells.Methods The coding?sequence(CDS) of TTF-1 gene was cloned into pCDH-CMV-MCS-EF1-CopGFP-T2 A-puro lentiviral vectors,which construct lentiviral vectors with TTF-1 overexpression. By packaging within 293 T cells,lentiviral particles with TTF-1 gene overexpression were obtained and transfected into lung adenocarcinoma cell line A549. An iCelligence real-time label-free cell function analyzer was used to detect A549 cell proliferation levels in TTF-1 transfection group(LV-TTF1),empty vector group(vector) and non-transfection group(NC). After stimulated by TGF-β1,the morphologic changes of A549 cells were observed. The RT-PCR method was used to detect the changes of EMT-related marker. Cell scratch test and Transwell migration assay were used to detect the changes of cell migration capacity.Results RT-PCR and Western Blot results showed that A549 lung adenocarcinoma cell line with TTF-1 overexpression was successfully constructed. ICelligence results showed that LV-TTF1 group had low cell proliferation level compared with vector and NC groups. The cells in LV-TTF1 group stimulated by TGF-β1 did not show significant changes in EMT morphology,whereas the changes were obvious in vector and NC groups. The expression level of E-cadherin mRNA in cells of LV-TTF1 group was significantly higher than that in NC group,and the expression level of Vimentin mRNA was significantly lower(P<0.05);the cell migration rate in LV-TTF1 group was smaller than NC group in scratch test(P<0.05). and the cell migration number in LV-TTF1 group was significantly less than that in NC group(P<0.05) in Transwell migration test.Conclusion TTF-1 overexpression mediated by lentiviral vector can inhibit the proliferation and EMT of lung adenocarcinoma A549 cells.